Fig 1: Significance of GAG attachment domain of SRGN in its promotion of ESCC cell invasion, ERK activation and c-Myc upregulation. (A) Effects of forced expression of intact SRGN, SRGN with truncated GAG (∆GAG), and mutated GAG (mGAG) on invasion (left panel) and migration (right panel) of ESCC cells. (B) Transwell invasion assay showing the effects of the CM collected from KYSE150-SRGN cells as chemoattractant on invasion of KYSE150 and KYSE410 cells, compared with CM from ∆GAG, mGAG, and CON cells. (C) Heatmap of reverse phase protein array (RPPA) analysis performed on cell lysates of four ESCC cell lines with SRGN overexpression compared with their corresponding control cells. The red bars on the right side indicate the proteins associated with the MAPK pathway. (D) Western blotting analysis of MEK/ERK pathway cascade and c-Myc in ESCC cells expressing SRGN, ∆GAG, mGAG, and CON. (E) Western blotting of subcellular fractions (cytosol and nuclear) of ESCC cells with SRGN, ∆GAG, mGAG overexpression. (F) Effect of SRGN-knockdown on MEK/ERK pathway cascade and c-Myc expression in ESCC cells. (G) Comparison of c-Myc expression in tumor xenografts established from SRGN-knockdown cells and vector control cells.
Fig 2: P53 transcriptionally upregulates the midkine cytokine in glioma cells.(a) The heatmap of ChIP-seq data using an antibody against p53 protein. The ChIP-seq data were obtained from the GSM2944126, GSM2944127, GSM2296271 and GSM2296277 datasets. (b) Snapshots of the UCSC genome browser showing the p53 ChIP-seq data at the locus of the MDK gene. Site1 and site2 were predicted by the JASPAR database, and site3 was used as a negative control. (c-d) The ChIP-qPCR assay and the luciferase reporter assay showed that p53 binding in the promotor of the MDK gene was higher in LN229R and HG7R cells compared with the LN229 and HG7 cells. Error bars indicate mean ± SD. ** P < .01; Student's t-test. (e) Circos plots showed the RNA expression levels of TP53 and MDK in p53-NC and p53-siRNA cells. (f-g) Relative mRNA expression level and protein expression level of MDK. In TP53 knockdown groups, the MDK mRNA expression level and protein expression level was lower than those in si-NC group.
Fig 3: Midkine (MDK) and follistatin-like 3 (FSTL3) are upregulated specifically in the lcSSc-PAH population. Graphs show the differential expression of MDK and FSTL3 relative to healthy controls with a side-by-side comparison to patients with dcSSc, SSc-ILD. Data is displayed as log2 expression levels and comparisons made by ANOVA: corrected with Bonferroni’s multiple comparison test. dcSsc diffuse systemic sclerosis HC healthy control, ILD interstitial lung disease, lcSSc-PAH limited cutaneous systemic sclerosis pulmonary arterial hypertension
Fig 4: Significance of ERK pathway in SRGN-induced cell invasion and c-Myc stabilization. (A) Effects of SRGN-knockdown on mRNA expression of SRGN, CD44, c-Myc, CCND1 in ESCC cells. (B) SRGN-overexpressing ESCC cells were treated with trametinib (at indicated concentrations) for 24 h, and the SRGN mRNA expression level evaluated by RT-PCR. Cells treated with DMSO served as control. (C) SRGN-overexpressing ESCC cells were treated with 100 nM trametinib for 48 h, and then evaluated by invasion assay (upper panel, scale bar, 100 µm). The quantification is shown in the lower panel. (D) SRGN-overexpressing cells were treated with increasing concentrations of trametinib as indicated for 72 h. The expressions of members of the MEK/ERK pathway cascade and c-Myc were evaluated by western blotting. Untreated vector controls were included for comparison. (E) KYSE150-SRGN cells with or without trametinib (100 nM) treatment were incubated in 100 µg/mL cycloheximide (CHX) for indicated duration before western blotting analysis (left panel). The numbers below the c-Myc blots are the band intensities of c-Myc that were normalized against GAPDH and then expressed relative to that at 0 h time point. The relative c-Myc degradation rate is presented in the graph (right panel). (F) KYSE150 cells with SRGN-knockdown were subjected to CHX chase assay and the band intensities of c-Myc were quantified. CHX chase assay results of KYSE410 cells are shown in Figure S6A-B.
Fig 5: SRGN upregulates and binds to MMP2 and MMP9. (A) Effect of SRGN, ∆GAG, mGAG overexpression on the expression of MMP2 and MMP9 in cell lysates and CM. (B) Effect of SRGN-knockdown on the expression of MMP2 and MMP9 in cell lysates and CM. (C) Western blot analysis of MMP2, MMP9, MDK in cell lysates and CM of SRGN-overexpressing cells treated with increasing concentrations of trametinib for 72 h. Untreated vector control cells were included for comparison. (D) Representative images of KYSE150 cells immunostained for SRGN (red) and MMP2 or MMP9 (green) are shown in the left panel. Scale bar, 10 µm. Line-scan profiles of immunofluorescence signals along the white arrows were acquired which showed co-localization between SRGN and the two MMPs (right panel). (E) Representative PLA images (with DAPI counterstain) confirmed the interaction between SRGN and MMP2/MMP9 in KYSE150 cells (white arrows). Negative control was conducted by replacing MDK antibody with rabbit IgG. The images of another cell line KYSE410 are presented in Figure S10B. (F) Whole cell lysates of MMP2-SFB (left panel) and MMP9-SFB (right panel) transfected cells were immunoprecipitated by anti-FLAG M2 beads before immunoblotting for SRGN. Western blotting showed that glycosylated SRGN at ~250 kDa co-precipitated with MMP2 and MMP9 (red frames), whereas the core protein of SRGN at ~28 kDa co-precipitated with MMP9 (yellow frames) but not with MMP2 (yellow frames with dashed lines).
Supplier Page from Abcam for Human Midkine ELISA Kit